In recent work, we have found that, under solution conditions, several nitroxide-labeled analogs of phenylalanine and tryptophan all exhibit specific binding to hemoglobin (Hb) at a region close to the beta-chain N-termini -- a location that was not observed in previous x-ray co-crystallization studies by others. Quantitative structure-activity relationship (QSAR) analysis of a series of ring-modified phenylalanine and tryptophan analogs that inhibit sickle hemoglobin (HbS) aggregation also indicates that the indole exhibits significant stereospecificity in binding to Hb; bromination at the 5-position substantially increases activity of the amino acid alone or incorporated at specific positions in dipeptides. In this project, we propose to build on this initial foundation, and to utilize a systematic structurally based approach for the design of antisickling agents with high inhibitory activity, and high specific affinity for hemoglobin. The basic goals of this project are to experimentally determine the detailed binding site location and binding stereochemistry for aromatic inhibitors of HbS gelation through high field nuclear magnetic resonance (NMR) measurements, to utilize theoretical predictions based on distance geometry analysis of known inhibitors and molecular dynamics sampling of binding regions to refine the binding site location, to design improved inhibitor structures with enhanced Hb binding affinity and specificity over serum albumin using computer-aided molecular modeling, to develop practical routes for their synthesis, and, if needed, to design modifications to permit ready erythrocyte membrane traversal. We anticipate an iterative process in which the data from experimental studies will be used as a base for the design of new inhibitors; the best predictions will be synthesized and tested, and succeeding cycles of refinement will proceed until an inhibitor with appropriate antigelation activity, specific hemoglobin affinity and membrane permeability is developed.